In 1522, Milan once again changed hands in dramatic fashion. The duchy continued to serve as a bellwether for the wider changes occurring in the Italian Wars and Mediterranean politics, with its fall to Imperial forces marking a turning point in the conflict and signalling a shift in the balance of power. On April 27, the Spanish infantry led by Prospero Colonna defeated the Franco-Venetian army at the Battle of Bicocca, just north of Milan. The governor of Milan, Odet de Foix, was defeated by the imperial army's combination of traditional pike and shot formations, and new tactics involving the use of wagons to protect their flanks. The triumph led to the collapse of French control in northern Italy, allowing for the imperial capture of Milan and Genoa. It was a decisive victory for the twenty-two-year-old Charles V against his rival, Francis I. Buoyed by the victory, the emperor elect claimed that the remaining Italian states were part of his empire, and they should pay to maintain the imperial army in Lombardy. Charles's agents extorted money from Siena, Lucca, and Florence, although the states claimed the money was gifted and not a regular payment.

The Battle of Bicocca marked the beginning of French decline in Italy and the rise of the Habsburgs in Europe. Charles's victory at the Battle of Bicocca intensified the nascent rivalry between Francis and Charles, and confirmed the permanent shift in the status quo in Italy.